Intestinal crypt cell survival plays a crucial role in conditions that affect cell turnover as a consequence of their function as the proliferative unit for the intestinal epithelium. Understanding the specific mechanisms that these cells use to inhibit programmed cell death, or apoptosis, will aid in the treatment of diseases in which there is enterocyte loss, such as the short gut syndrome. Regulation of the processes that determine whether a crypt cell undergoes apoptosis or survives relies, in part, on extracellular signals that activate second messengers such as adenosine 3', 5'-cyclic monophosphate (cAMP). Preliminary studies have shown that in the undifferentiated T84 intestinal cell line, cAMP inhibits apoptosis. In addition, we have demonstrated that cAMP activates a known survival pathway in these cells, the extracellular-signal regulate kinase (ERK1/2) cascade. This application will address the hypothesis that cAMP inhibits apoptosis in undifferentiated intestinal epithelial cells through the activation of the ERK1/2 signaling cascade. We propose the following specific aims: 1.) elucidation of the signal transduction pathway involved in cAMP-mediated initiation of ERK1/2 signaling, 2.) establishment of the ERK1/2 cascade as a mechanism for cAMP-induced inhibition of apoptosis, and 3.) the in vivo demonstration of cAMP-induced inhibition of intestinal apoptosis. In vitro studies will use a combination of pharmacological agents and molecular inhibitors to dissect two critical aspects of cAMP-mediated ERK1/2 activation, namely the activation of a cAMP responsive binding protein and the phosphorylation of a Raf kinase isoform at the entrance point of the ERK1/2 cascade. In addition, we will determine the specific survival pathways induced by cAMP to inhibit apoptosis. The in vivo studies will use a mechanistic approach to reverse irradiation induced crypt cell apoptosis by increasing intestinal levels of cAMP and demonstrate the role of cAMP in the protective effects of prostaglandin E2 signaling. These studies will lay the ground work for utilizing cAMP-mediated signaling to enhance intestinal proliferation at the level of the crypt cell and to enhance the adaptive response in short gut syndrome and in other disorders of enterocyte loss.